Identification of essential survival genes

ABSTRACT

A method for identifying a strain having a conditional lethal mutation which is essential for survival when strain is incubated under restrictive growth conditions, and methods of identifying gene products and gene functions thereof.

BACKGROUND OF THE INVENTION

The invention relates to methods of identifying genes and correspondinggene products which are required for survival.

SUMMARY OF THE INVENTION

The invention features a method for identifying a strain carrying alethal conditional-sensitive mutation in a gene essential for survival.The method includes (a) growing organisms (e.g., cells) under firstpermissive conditions; (b) exposing organisms from step (a) torestrictive conditions for a period of time equivalent to at least twogrowth cycles (e.g., cell cycles); and (c) shifting the organisms fromstep (b) to second permissive conditions for a period of time equivalentto at least ten growth cycles (e.g., cell cycles). Following thistreatment, mutant organisms which both (i) failed to grow when exposedto the restrictive conditions of step (b), and (ii) failed to resumegrowth when returned to the second permissive conditions of step (c) areselected (step (d)).

This selection process separates strains having lethal mutations fromstrains having nonlethal or static mutations. In general, a selectedstrain has a gene that is sensitive to the restrictive conditions andthat is essential for growth. The transient shift to a restrictivecondition results in loss of the gene product; this loss is lethal tothe organism. For example, the gene product of the mutated gene is notfunctional under the restrictive conditions.

Another aspect of the invention features a method for identifying anessential gene in a strain. This method includes (a) growing organisms(e.g., cells) under first permissive conditions; (b) exposing organismsfrom step (a) to restrictive conditions for a period of time equivalentto at least two cell cycles; (c) shifting the organisms from step (b) tosecond permissive conditions for a period of time equivalent to at leastten cell cycles; (d) selecting a strain having a lethal mutation,wherein the strain (i) failed to grow when exposed to the restrictiveconditions of step (b), and (ii) failed to grow when shifted to thesecond permissive conditions; (e) identifying a strain selected in step(d) which carries a recessive conditional lethal mutation; and (f)identifying from the strain of step (e) a gene corresponding to the geneencoding the recessive conditional lethal mutation. Preferably, failureto grow is defined by at least three logs of killing, i.e., if 1000cells are grown under permissive conditions, shifted to restrictiveconditions, and then shifted to second permissive conditions,statistically 999 cells are dead. This threshold is intended to identifythe more sensitive conditional mutations. Failure to grow can also bedefined as 1.5, 2, 3.5, 4, or 5 logs of killing.

Another aspect is a method for identifying a gene product target of abiocidal drug (e.g., antimicrobial, antiparasitic, or insecticidal),including the steps of: (a) growing strains (e.g., fungal, bacterial,parasitic, or insect) under first permissive conditions; (b) exposingthe strains from step (a) to restrictive conditions for a period of timeequivalent to at least two growth cycles; (c) shifting the strains fromstep (b) to second permissive conditions for a period of time equivalentto at least ten growth cycles; (d) selecting a strain having a genecarrying a conditional lethal mutation; and (e) identifying the geneproduct corresponding to the conditional lethal mutation, therebyidentifying a gene product target of a biocidal drug.

Once a mutant organism (strain) is identified, routine techniques may beused for transformation, amplification, isolation, purification, andsequencing the gene carrying the mutation. Essential survival genes arerequired for growth (e.g., metabolism, division, or reproduction). Suchgenes and gene products are useful in developing therapeutic agents suchas antifungal, antibacterial, and antiparasitic agents; insecticidalagents; and preventive antimicrobial agents. Therapeutic agents canreduce or prevent growth, or decrease pathogenicity or virulence, andpreferably, kill the organism. The genes and gene products identified bythe invention can also be used to develop antimicrobial agents which areeffective in preventing microbial infection, e.g., by inhibiting theestablishment of a bacterial biofilm, in addition to agents which areuseful in the treatment of an established infection.

Other features and advantages of the present invention will be apparentfrom the following detailed description of the invention, the examples,and also from the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention features a method of identifying mutant organisms havingconditional-sensitive lethal mutations, and subsequently gene productsthereof. The disclosed methods are useful for high-throughput (e.g., useof 96-well plates) screening of genomic or mutant libraries to rapidlyidentify genes, and corresponding gene products, which are essential forsurvival. By altering restrictive conditions, including incubationperiod, temperature, concentration of an antibiotic, a salt, pH, and soon, and by altering the threshold level of "lethal" (how many logs ofkilling), the strains can be prioritized.

The selected strains cannot survive under restrictive conditions. Theeffect of a lethal mutation cannot be reversed or overcome by shiftingthe organisms to permissive conditions. The selected genes and productsthereof are therefore essential for survival of the organism underrestrictive conditions. In contrast, strains having nonlethal or staticmutations may grow very slowly or even appear inactive under restrictiveconditions. The effect of a static mutation is reversible; organismshaving static mutations resume metabolism and growth when shifted topermissive conditions. Selection of a conditional-sensitive lethalmutant organism allows identification of the gene carrying the lethalmutation and identification of the corresponding gene product, if any.

A conditional lethal mutation results in a gene or a protein which isnot functional under restrictive conditions. A non-functional gene canhave a defect in the promoter resulting in reduced or abnormal geneexpression. A non-functional protein may have a conformational defectcausing improper protein folding or abnormal protein degradation.Improper protein folding can result in partial or total failure to fold,to recognize a native substrate, and/or to bind and release thesubstrate.

Therapeutic agents can be developed from the identification of essentialgenes of organisms such as bacteria or fungi. Preferably, a gene product(e.g., a protein or an RNA molecule) identified by the methods disclosedherein is distinct from the gene products targeted by existing drugssuch as antibiotic or antifungal agents. The disclosed gene selectionmethods establish that the gene product is essential for survival of theorganism. Such an identified gene product therefore serves as a noveltarget for therapeutics based on a mechanism which is likely distinctfrom the mechanisms of existing drugs. Similarly, distinct from knowncompounds is a compound which inhibits the function of a gene productidentified by methods disclosed herein, for example, by producing aphenotype or morphology similar to that found in the original mutantstrain.

According to one aspect of the invention, a mutant collection issystematically screened to identify genes and preferably gene productswhich are targets for drugs. For example, an antimicrobial (e.g.,antibacterial or antifungal) drug may act as a biocide by bindingreversibly, or preferably irreversibly, to the identified gene or geneproduct target, and thereby impairing its function. Loss of the function(or the synthesis or the complete processing) of the gene product targetwill result in inhibition of microbial growth, and preferably willresult in death of the microbe. This aspect includes a method foridentifying biocidal agents, including the step of exposing a geneproduct corresponding to the wildtype sequence of a mutant sequenceidentified by methods disclosed herein to the test agent; and selectingagents which impair (preferably, selectively) the function of the geneproduct. The selection may be based on routinely measured parameterssuch as a binding constant, dose-response curves or other measurementsof inhibition and binding.

In one aspect, the restrictive conditions include a temperature at ornear the body temperature of a healthy or infected mammal (e.g., human).In this case, the selected genes are essential for survival of, forexample, a fungus at or near the temperature of the human body. In oneaspect of the invention, the mutations are temperature sensitive (ts).After identification of a strain carrying the lethal mutation, thelocation, function, and sequence of the gene and corresponding proteincan be determined. Elucidation of the mechanism(s) of action for thesetargets provides a rational basis for the design of therapeutic agentswhich are lethal or cidal to the cell used in the method, and relatedcell types.

In one embodiment of the method for identifying a gene product target ofa biocidal drug, the restrictive conditions include changing thetemperature and the conditional lethal mutation is atemperature-sensitive mutation or a cold-sensitive mutation. In general,the method is used for large-scale screening, and therefore includes atleast 50 or 75 strains, and preferably multiples of 96 strains in steps(a)-(c) through use of 96-well plates.

A. Lethal Mutations

The invention is based, in part, on the recognition that there are atleast two types of strains (e.g., cells) which fail to grow underrestrictive growth conditions: (a) strains with lethal mutations, and(b) strains with nonlethal or static mutations. In general, strains withlethal mutations are of greater interest for therapeutic researchbecause a bactericidal drug, for example, is generally more desirablethan a bacteriostatic drug.

According to the invention, strains carrying lethal mutations mustsatisfy two criteria. First, when exposed to restrictive conditions forat least two growth cycles, the organism fails to grow. Examples of atleast two growth cycles include at least 3, 4, 5, 7, 8, 10 or moregrowth cycles. Measurements of growth include RNA synthesis, DNAsynthesis, protein synthesis, membrane morphology, success of divisionor reproduction, and levels of ATP. Examples of failure to growtherefore include serious membrane deformity, reduction or absence ofDNA (or RNA or protein) synthesis, lysis, ATP depletion, unsuccessfuldivision or reproduction, or even organism death.

The second criterion is that shifting such an organism from restrictiveconditions to second permissive growth conditions for a period of timewill not revive the organism or restore growth. This organism carries atleast one lethal mutation, the product of which is irreversiblysensitive to the restrictive conditions. The gene carrying this mutationis essential for growth during the incubation period under therestrictive conditions.

According to one aspect of the invention, a strain carrying a nonlethalor static mutation is expressly avoided, i.e., not selected. A strainwith a nonlethal or static mutation may fail to grow and reproduce underrestrictive conditions, and yet will resume growth when shifted fromrestrictive to second permissive growth conditions. This resumption ofsubstantially normal growth is generally apparent after two or moregrowth cycles under permissive conditions. In some cases, metabolism orgrowth may be initially slow during a transition period; in addition,growth may be slower than normal for several growth cycles. In eithercase, a strain with a static mutation does not satisfy the secondcriterion for lethal mutation as used herein.

Strains are shifted to the second permissive growth conditions for aperiod of time sufficient to distinguish the lethal mutations from thestatic or nonlethal mutations.

The period of time will vary with the method of detecting growth ordeath, but is generally equivalent to a plurality of growth cycles(e.g., at least 2, 4, 6, 8, 10, 15, or 20 cycles). Depending on theorganism and the difference between the restrictive and permissiveconditions, growth may be delayed, or the rate of growth may increaseduring a transition period before stabilizing. Growth can be measured bymethods known to those in the art, including expansion of colony cellmass, increased turbidity of a liquid cell culture suspension, cell ororganism staining, DNA synthesis, and protein synthesis.

One aspect of the invention provides a method for identifying lethalmutations which produce proteins that are functional at permissivetemperatures. Permissive conditions are any conditions under whichmutant growth (or growth rate) is at least about 75% of wildtype growth(or growth rate) under the permissive conditions.

In general, under restrictive conditions the wildtype growth rate is notless than about 25%, and preferably not less than about 50%, of thewildtype growth rate under permissive conditions. In bacteria,restrictive conditions include a temperature, wherein the differencebetween the first permissive and restrictive temperatures is between 5°and 20° C., and preferably between 5° and 15° C. According to thepresent invention, a temperature sensitive lethal mutation in fungalorganisms is a mutation in a gene that is required for fungal growth attemperatures between 5° and 15° C., and preferably between 5° and 12°C., different from a permissive temperature. Once exposed totemperatures several degrees different, (e.g., at least 5, 7, 8 or 10°C. higher or lower) than the permissive temperature (e.g., within a fewdegrees of optimal growth temperature) for at period at least two cellcycles, a strain with a conditional ts lethal mutation will not resumegrowth when shifted to permissive conditions.

B. Conditional Mutations

Conditional mutations are mutations wherein the protein cannot functionnormally (e.g., bind to or release from a substrate) under restrictiveconditions. In one aspect, the genes carrying recessive conditionalmutations which produce conformational changes in the gene productsthereof are preferred as therapeutic targets. This aspect provides amethod including the step of selecting a strain carrying a recessiveconditional lethal mutation.

Null mutations result in elimination of a gene product, as demonstratedby inability of the strain to survive under restrictive conditions.Dominant lethal mutations produce proteins which can bind a ligand, suchas another protein, under restrictive conditions but which are unable torelease the ligand. Transformation of a bacterial library of wildtypeDNA allows identification of recessive and dominant mutations. Recessivemutations were rescued by a clone which complemented the mutation, andtherefore allowed the strain to grow at restrictive temperatures.Strains carrying dominant mutations cannot be rescued by transformationand fail to grow at restrictive temperatures. In fungi, isolation of adiploid or partial diploid organism containing a wildtype allele as wellas the lethal mutation allows determination of whether the mutation isrecessive or dominant.

C. Prioritization, Complementation, and Gene Function

The disclosed methods efficiently distinguish cells carrying lethalmutations from cells carrying nonlethal or static mutations.Identification of lethal mutations leads, through a variety of paths, toefficient identification of the corresponding genes and gene products.The desired conditional mutations are then mapped and/or sequencedaccording to any of several methods known to those in the art ofmolecular biology. Once the strain is selected, conventional methods oftransformation, amplification, isolation, and sequencing are used toidentify the gene and determine the sequence. Conventional methods areused, preferably, in combination with any of the methods described inthe next paragraph or in the Examples.

For example, one embodiment includes the further step of isolating froma selected strain a gene carrying the mutation. Other embodimentsinclude the further step of identifying the function of a gene carryingthe mutation, and the further step of sequencing a gene carrying themutation, respectively.

Where complementation involves DNA having multiple genes, transposonmutagenesis can identify an individual gene which complements the tsmutation (Example 4). Genes can also be prioritized by identifying whatgene function or phenotype the mutation affects, such as DNA synthesis,RNA synthesis, protein synthesis, protein secrection, or cell envelopeintegrity (Example 5). The genes identified by the disclosed methods canbe subcloned from plasmid, cosmid, or phage gene libraries by geneticcomplementation. The disclosed methods are suitable forindustrial-scale, high-throughput screening of gene libraries.

D. Organisms

Turning to the first step (a) of growing strains under permissiveconditions, in principle, any type of organism or cell which is haploidcan be used. Non-haploid organisms susceptible to homozygous mutation(e.g., arabidopsis) can also be used. When speed is desirable, organismsor cells which can be plate cultured are preferred. Organisms can beobtained from a symptomatic or asymptomatic host or from naturalenvironments (e.g., cell colonies growing on decomposed organicmaterial, airborne cells, and cells trapped in ice or found in aqueoussolutions). Hosts include plants (e.g., food crops, trees, or fibercrops) and animals, such as mammals (e.g., humans) or domestic animals(horses, cows, pigs, poultry, cats, dogs, mice, rats), birds, fish, andamphibians. Organisms used in the method include bacteria, fungi, yeast,nematodes (e.g., C. elegans), protista (e.g., tetrahymena andparamecium), Rama pipiens, and Drosophila spp.

Bacterial strains include Gram-positive cocci such as Staphylococcusaureus, Streptococcus pyogenes (group A), Streptococcus spp. (viridansgroup), Streptococcus agalactiae (group B), S. bovis, Streptococcus(anaerobic species), Streptococcus pneumoniae, and Enterococcus spp.;Gram-negative cocci such as Neisseria gonorrhoeae, Neisseriameningitidis, and Branhamella catarrhalis; Gram-positive bacilli such asBacillus anthracis, Bacillus subtilis, Corynebacterium diphtheriae andCorynebacterium species which are diptheroids (aerobic and anerobic),Listeria monocytogenes, Clostridium tetani, Clostridium difficile,Escherichia coli, Enterobacter species, Proteus mirablis and other spp.,Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, Shigella,Serratia, and Campylobacter jejuni. Bacterial infections result indiseases such as bacteremia, pneumonia, meningitis, osteomyelitis,endocarditis, sinusitis, arthritis, urinary tract infections, tetanus,gangrene, colitis, acute gastroenteritis, bronchitis, and a variety ofabscesses, nosocomial infections, and opportunistic infections.

Fungal organisms include dermatophytes (e.g., Microsporum canis andother M. spp.; and Trichophyton spp. such as T. rubrum, and T.mentagrophytes), yeasts (e.g., Candida albicans, C. Tropicalis, or otherCandida species), Saccharomyces cerevisiae, Torulopsis glabrata,Epidermophyton floccosum, Malassezia furfur (Pityropsporon orbiculare,or P. ovale), Cryptococcus neoformans, Aspergillus fumigatus,Aspergillus nidulans, and other Aspergillus spp., Zygomycetes (e.g.,Rhizopus, Mucor), Paracoccidioides brasiliensis, Blastomycesdermatitides, Histoplasma capsulatum, Coccidioides immitis, andSporothrix schenckii. Fungal infections (mycoses) may be cutaneous,subcutaneous, or systemic. Superficial mycoses include tinea capitis,tinea corporis, tinea pedis, onychomoycosis, perionychomycosis,pityriasis versicolor, oral thrush, and other candidoses such asvaginal, respiratory tract, biliary, eosophageal, and urinary tractcandidoses. Systemic mycoses include systemic and mucocutaneouscandidosis, cryptococcosis, aspergillosis, mucormycosis (phycomycosis),paracoccidioidomycosis, North American blastomycosis, histoplasmosis,coccidioidomycosis, and sporotrichosis. Fungal infections alsocontribute to meningitis and pulmonary or respiratory tract diseases.Opportunistic fungal infections have proliferated, particularly inimmunocompromised patients such as those with AIDS. Preferred organismsinclude Escherichia coli, Streptococcus pneumoniae, Staphylococcusaureus, Saccharomyces cerevisiae, Aspergillus fumigatus, and Aspergillusnidulans. See Goodman and Gilman's Pharmacological Basis ofTherapeutics, (8th ed., 1990) Table 44-1, page 1024-1033, for additionalmicrobial pathogens, diseases, and current therapeutic agents. Theabove-described cells are generally available, for example, from theAmerican Type Culture Collection.

E. Mutagenesis

Whatever their source, cells can be mutagenized. Mutagens induce changesin DNA, by acting on one or more bases or by being incorporated into thenucleic acid. In bacteria, the kill rate is preferably 90% (10% or lesssurvive). In yeast and fungi, it is preferable to have a survival rateof 20-50% (e.g., 30%) when mutagenized cells are grown (e.g., replicaplated) because cells viable under permissive conditions are necessaryfor cloning those strains which carry the desired lethal mutations.

Chemical mutagens include ethylmethanesulfonate (EMS),methylmethanesulfonate (MMS), methylnitrosoguanidine (NTG),4-nitroquinoline-1-oxide (NQO), 2-aminopurine, 5-bromouracil, ICR 191and other acridine derivatives, sodium bisulfite, ethidium bromide,nitrous acid, hydroxylamine, N-methyl-N'-nitroso-N-nitroguanidine, andalkylating agents. Physical mutagens include ultraviolet radiation andx-rays.

Mutagenesis is accomplished according to methods well-known in the art(see, e.g., Current Protocols in Molecular Biology 1995, Vol. 2, Section13.3, wherein all the cited references are dated 1990 or earlier).Conditions for mutagenesis such as concentration (chemical mutagenesis)or intensity (e.g., ultraviolet mutagenesis) and duration are preferablyoptimized to produce a high rate of mutation while minimizing the amountof killing among the exposed cells. In general, mutagenesis is performedat a temperature that is below the optimal growing temperature for thattype of organism, because the sub-optimal temperature has been found todecrease cell killing. For example, survival curves can be plotted witha constant time of exposure and varying concentrations of mutagen, or avaried time of exposure and a constant concentration of mutagen. See,e.g., Adelberg et al., Biochem. Biophys. Res. Comm. 18:788 1965. Killcurves or survival curves are suggestive of mutagenic frequency. Optimumconcentrations of nitrosoguanidine have been reported for Haemophilusinfluenzae, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa,Staphylococcus aureus, and Listeria monocytogenes as, respectively, 2,3, 10, 20, 10-20, and 50 (μg/mL) (see, e.g., Morris Hooke, et al., Meth.Enzymol. 34:448, esp. Table I at 451).

F. Permissive and Restrictive Growth Conditions

Growth conditions include temperature, pH, type and concentration ofcarbon and nitrogen sources, trace minerals, vitamins, salts, cellextracts, proportion of oxygen and carbon dioxide, humidity, presence orabsence of conidia-forming materials such as DMSO, glycerol, anddeuterated water, and presence or absence of osmotic stabilizers such assucrose and potassium chloride.

Embodiments of the invention include the following limitations,individually or in combination. Permissive conditions include: acomplete medium for the cells to eliminate mutants that are defective innutritional requirements such as amino acid biosynthesis; and a mediumat low osmotic strength to eliminate mutants with defective cell wall orcell surface mutations. In general, permissive conditions should allowstrain growth or a growth rate that is at least 75% of that of thewildtype growth or growth rate.

The second permissive conditions can be the same as, or different from,the first permissive conditions. The period of time under the secondpermissive conditions can be equivalent to at least 2, 5, 10, 15, or 20growth cycles or more. It is important that the period be long enough toallow organisms carrying irreversible lethal mutations to bedistinguished from organisms carrying reversible static mutations. Thelatter may require time to resume growth after shifting to the secondpermissive conditions.

Restrictive conditions reduce or arrest growth. Restrictive conditionsare non-optimal (above or below optimal) growth conditions. Preferably,restrictive conditions are sufficient to affect the function of mutatedgenes or gene products. For bacteria, a temperature between 5° and 25°C. (or between 10° and 15° C.) below the optimal growth temperature forwildtype; for fungi, a temperature between 5° and 15° C. (or between 5°and 12° C.) below the optimal growth temperature for wildtype. Ingeneral, restrictive conditions for bacteria should result in straingrowth that is at least 50% growth or growth rate of the wildtype.

Optimal temperatures are 30° C., 37° C., and 37° C. for S. cerevisiae,A. nidulans, and A. fumigatus, respectively; sample restrictivetemperatures which are higher than optimal growth temperatures are 36°C., 42° C., and 42° C., respectively. The restrictive incubation periodshould be at least 2 growth cycles in length, and generally not morethan 70 growth cycles. For many microbes, the incubation is 3, 4, 5, 7,or 10 cell cycles, but not more than 24 hours.

Restrictive conditions include a temperature between the optimal growthtemperature for the cells and 15° C. above the optimal growthtemperature for the cells. Restrictive/permissive condition pairsinclude high/low salt concentrations, high/low temperature, low/hightemperature (cryosensitive mutations), high/low osmotic pressure,low/high osmotic pressure, aerobic/anerobic incubation, anerobic/aerobicincubation, glycerol/no glycerol, no DMSO/DMSO, deuterated water/nodeuterated water, no deuterated water/deuterated water, low/high pH, andhigh/low pH.

G. Growth or Failure to Grow Under Restrictive Conditions

According to the invention, the transient disruption of gene functionduring the period of restrictive growth conditions results in celldeath. Organism death can be macroscopically observed in a colony whichhas the same or reduced size over several growth cycles under the secondpermissive conditions. Light microscopy and staining can revealcytological deformations or other morphologies known by those in the artto be indicative of cell death. Under permissive or at least optimalconditions, protein synthesis occurs in a cell which is nominally alive.Dead cells are characterized, in part, by lysis or the absence of DNA,RNA, and protein synthesis.

Note that "temperature sensitive mutants" as used by others includesmore than one type of mutant. For example, one type of mutant has alesion in a gene which is turned on by heat shock and has an essentialfunction turned on during the transition to a restrictive temperature.This type of mutant may survive exposure to temperatures higher than theoptimal growth temperature. Another type of ts mutant is affectedreversibly by exposure to restrictive temperatures. Neither of these twotypes is a temperature sensitive lethal mutation. Further guidance isprovided by the Examples below.

EXAMPLES Example 1

Temperature Sensitive Escherichia coli Mutants

Log phase E. coli cultures (ATCC # K802) were mutagenized in LB mediumat 37° C. without aeration. Four incubation treatments were used: (i)EMS (10 μL/mL) for 120 minutes, (ii) NQO (20 μg/mL) for 60 minutes,(iii) NTG (30 μg/mL) for 15 minutes, and (iv) NTG (10 μg/mL) for 30minutes. After treatment, cells were washed and resuspended in fresh LBmedium and grown overnight at 30° C. LB medium consists of 10 gbactotryptone, 10 g sodium chloride, and 5 g yeast extract brought to 1L with distilled water.

The overnight culture was diluted 100-fold into fresh LB medium andgrown at 30° C. to log phase. After ampicillin (100 μg/mL) was added tothe log phase culture, the culture was incubated at 42° C. withoutaeration for 2 hours. Cells were washed and resuspended in fresh LBmedium and grown overnight at 30° C.

After 10⁷ -fold dilution in LB medium, 100 μL aliquots of culture werespread onto LB agar plates and incubated overnight at 30° C., which islower than the optimal growth temperature. LB agar plates were preparedwith LB medium and 15 g of bactoagar per liter of LB medium.

Each plate (100-200 cfu/plate) was replicated onto another LB agar platewhich was then incubated at 42° C. for 7 hours. The master plates wereincubated at 30° C. for 7 hours. Colonies which grew well at 30° C. butgrew poorly or not at all at 42° C. were struck onto duplicate LB agarplates. The phenotype was confirmed by incubating one duplicate plate at30° C. and another duplicate plate at 42° C. overnight, and comparingthe colonies again. A total of 1500 strains in minimal medium wereisolated on LB medium. Twelve genes were isolated and sequenced, one ofwhich has no previously known function. Seventeen genes were isolatedand sequenced from screening Saccharomyces cerevesiae, two of which haveno previously known function.

Example 2

Temperature Sensitive Aspergillus nidulans Mutants

Aspergillus nidulans (ATCC #FGSC4) were mutagenized following S. D.Harris, et al., Genetics 136:517-532 1994 using 4-nitroquinoline asmutagen.

1150 mutagenized strains were isolated following incubation for 16 hoursat 28° C. in minimal medium (MN) medium (pH 6.5, 1% glucose, nitratesalts and trace elements as described in the appendix of Kafer, Adv.Genet. 19:33-131, 1977). Trace element solution was stored at 4° C. inthe dark; each liter contained 40 mg Na₂ B₄ 0₇ (10 H₂ O), 400 mg cupricsulfate (5 H₂ O), 1 g ferric phosphate (4 H₂ O), 600 mg manganesesulfate (4 H₂ O), 800 mg disodium molybdate (2 H₂ O), and 8 g zincsulfate (7 H₂ O). Salt solution was stored at 4° C. after adding 2 mlchloroform as a preservative; each liter contained 26 g potassiumchloride, 26 g magnesium sulfate (7 H₂ O), 76 g monobasic potassiumphosphate and 50 mL trace element solution. Supplement solution issterilized by autoclaving for 15 minutes and stored in a light-proofcontainer due to reactivity of riboflavin. Each liter contains 100 mgnicotinic acid, 250 mg riboflavin, 200 mg pantothenic acid, 50 mgpyridoxin, 1 mg biotin, and 20 mg p-aminobenzoic acid.

Conidia (2×10⁶ /mL in sterile distilled water) were mutagenized with NQO(4 μg/mL) for 30 minutes at 37° C. with constant shaking. Diluting theconidia with an equal volume of 5% sodium thiosulfate inactivated theNQO. Mutagenized conidia were diluted and plated onto CM+TRITON˜ X-100plates (registered by Union Carbide Chemicals) and incubated at 28° C.for 3 days. Colonies were replica plated and the replica plates wereincubated at 28° C. and 42° C. Putative ts mutants were picked andretested, then stored as a colony plug in 15% glycerol at -70° C.

Cells were replica plated and shifted to 42° C. for 24 hours. Strainsthat grew poorly or not at all were selected. Out of 1150 originalstrains, 10 did not recover from the 42° C. incubation period.

These 10 strains were transformed with an Aspergillus genomic coslibrary in pCosAX vector (see Adams and Borgia et al., FEMS Microbiol.Lett. 122:227-231 1994. Strains were grown for 3-4 days at 28° C.,replica plated and shifted to 42° C. for a maximum of 3 days. Strainswhich grew were collected; cosmid was recovered from DNA of collectedstrains. The cosmid was packaged using GIGAPACK™ III Gold packagingsystem (Stratagene, La Jolla, Calif.) which produced a plasmid which wasisolated, purified, and used to transform bacteria for amplification,isolation, purification, and sequencing. Three genes were isolated andsequenced, two of which are known and one of which has no previouslyknown function.

Example 3

Temperature Sensitive Yeast Mutants

The Abelson library was mildly mutagenized in two sets (A and B)(available from CalTech, Pasadena, Calif.). Set A was SS328 MATαade2-101 ura3-52 his3 Δ200 lys2-800; set B was SS330 MATα ade2-101ura3-52 his3 Δ200 tyr1. The Hartwell library was heavily mutagenized(University of Washington); set C was A364a MATα ade-1 ade-2 uro-1 his-7lys-2 tyr-1 gal(-).

All clones were temperature sensitive and required exogenously supplieduracil for growth. Cells were grown in YPD medium containing 2% peptone,1% yeast extract, and 2% glucose in 1.8% agar at 26° C. for 2 days. Aclone was grown in a microtiter well at 37° C. for 5 hours, then shiftedto 26° C. for 16 hours. Cell growth was measured by reading at OD₆₅₀ nm.After ranking clones according to growth, clones in the lowest 35thpercentile were selected. Microscopic examination of these clonesresulted in the second round of selection based on irregular orsickly-looking morphology.

Using the master plate as a source, a low-growth clone was grown underosmotic stabilization conditions on YPD medium with 1M sorbitol.Twenty-two out of 99 Abelson clones (22%) were susceptible to sorbitol.

Example 4

Antibacterial Target Genes and Gene Products

a. Ts Mutant Isolation

Survival curves are prepared using nitrosoguanidine,ethylmethanesulfonate, or hydroxylamine. Temperature-sensitive mutantsare isolated on a large scale as follows.

Mutagenize cells and plate survivors at 30° C. Store mutagenizedculture. Ts mutants that cannot grow at 42° C. are enriched by selectionin the presence of a cell wall-active agent such as ampicillin byshifting culture to 42° C. for 2 hours, and returning cells to 30° C.Cells are replica plated at 42° C. and 30° C. overnight. Test a thickstreak of putative ts mutants at 42° C. and 30° C. Streak for singlecolonies from the 30° C. plate. Store the mutants. Prioritize mutants asdiscussed in Example 5.

b. Library Construction

Select a vector for cloning wild type DNA of the organism that wasmutagenized, and use a shuttle vector if mutagenesis is in an organismother than E. coli. The vector may contain a signal element whichindicates the presence of an insert, such as a blue-white selectionresulting from insertional inactivation of the lacZ gene. Prepare alarge wild type chromosomal DNA, such as a SauIIIA1 partial digestion of5-10 Kb inserts. Pool white transformants into 96-well trays if thereare fewer than about 3000 transformants (or scrape plates if more thanabout 10,000 transformants). Prepare pooled DNA and transform into E.coli, and the organism that was mutagenized if other than E. coli, suchas as S. aureus. To assure presence of inserts at a high frequency,prepare 24 minipreps of transformants from both organisms.

c. Complementation of ts Mutants

Transform ts mutants with genome library at 42° C. Include a controlplasmid without insert. Pool transformants and prepare DNA to create anenriched pool. Transform with enriched pool at 30° C. Replicatetransformants from 30° C. plate onto plates at 42° and 30° C. toidentify putative complemented clones. Streak candidates for singlecolonies from 30° C. plate on plates at 42° and 30° C. Make several(e.g., 5) sequencing grade plasmid preps from complemented clones.Transform the ts mutant, with the sequencing-grade plasmids at 30° C.Streak 5 transformants of each transformation at 42° and 30° C., andcompare to control to identify candidates for sequencing.

d. Redundancy of ts Mutants

Redundancy can be monitored by either method described below. For eachcollection of 10 ts mutants that have been complemented, make a pool ofcomplementing plasmid. Test each pool of 10 against subsequent tsmutants as they are isolated, or as soon as priority is established by akiller ts phenotype. Alternatively, test redundancy within a group of 10using 90 transformations. Make pools A-E of plasmids that complementmutants 1-10. Pool A contains plasmids complementing mutants 1-5; pool Bcontains plasmids complementing mutants 6-10; pool C contains plasmidscomplementing mutants 3-7; pool D contains plasmids 8, 9, 2, and 3; andpool E contains plasmids 1, 4, 6, 8, and 10. The mutants and thecorresponding pools for transformation are as follows: 1 (B, C, D); 2(B, C, E); 3 (B, E) will not detect 2 or 5; 4 (B, D) will not detect 1or 5; 5 (B, D, E); 6 (A, D) will not detect 7 or 10; 7 (A, D, E); 8 (A,C) will not detect 9 or 10; 9 (A, C, E); and 10 (A, C, D).

e. Transposon Mutagenesis

Transform into donor strain containing gamma-delta on F' but lackingresolvase. Mate transformants into a NalR resolvase+strain selecting forApR NalR KmR. Prepare pooled DNA and transform the pooled DNA into thets mutant. Isolate transformants that fail to complement. Preparesequencing-grade plasmid preps, 2 non-complementing plasmids/ts mutantsmight be optimal, followed by sequencing.

Tn 1000 mutagenesis using ZK1328 (CBK884) Δ{Δsrl-recA)306::Tn10Δtet}277/pIF200 pOX38(45 kb HindIII F fragment/conjugationproficient)::mγδ-1,Kan)/pXRD4043 (pACYC184-tnpA, cat, IPTG-inducibletransposase). The recipient strain is ZK1329(LW49)F⁻recA56,chr::γδ,NAl^(r). Transform donor strain ZK1328 with complementingplasmid of interest. Select for the antibiotic resistance of incomingplasmid, Cm(40 μg/ml)-resistance and Kan resistance (30 μg/ml). Grow onetransformant in 1 ml LB plus 1 mM IPTG at 37° C., no shaking, therebyinducing transposase in the strain and allowing for F-pilus expression.The culture should be grown to 5×10⁷ to 1×10⁸ cells/ml (barely turbid).Concurrently grow 1 ml of the recipient strain ZK1329 in LB to earlystationary phase (2×10⁸ cells/ml). Mix 0.5 ml donor cells with 0.2 mlrecipient cells in 50 ml culture tube. Incubate at 37° C. with noshaking for 30 minutes. Add 5 ml prewarmed LB plus 1 mM IPTG, incubate 3hours at 37°, no shaking. Plate 0.1 ml cells on LB plus 150 μg/ml Ampand 10 μg/ml Nal, thereby selecting against the donor strain. Pool thetransconjugants and extract plasmid. There should be about 100 to 1000transconjugants per plate. Transform original mutant with pool. Checkfor non-complementing clones (those that do not complement at 42° C.).Isolate plasmid from 2 non-complementing clones and sequence. Tn10 canbe used, in the alternative.

Example 5

Prioritization of ts Mutants

By matching an assay to a phenotype under restrictive conditions (e.g.,a restrictive temperature), the nature of the mutation can bedetermined. For example, growing cells at the restrictive temperaturefor 2, 6, and 24 hours and then shifting to the permissive temperaturedetermines viability after the temperature shift. This considers theirreversibility of the defect; a mutation that results in the loss ofviability after a brief shift to the restrictive temperature suggests agene having an ideal cidal (lethal) target gene product. Monitoring ofthe optical density of cultures at the restrictive temperature candetect gene products whose loss of function leads to cell lysis.Similarly, microscopic observation can detect filamentation and defectsin cell division. Lysis mutants can be examined further for osmoticstabilization. Previously unknown genes which are essential for theintegrity or biosynthesis of the cell wall are of great interest.

Precursor labelling studies are also useful. Incorporation of labelledthymine in a short pulse experiment (e.g., 30 minutes, 1 hour, and 6hours after temperature shift) investigates the arrest of DNA synthesis.In bacteria, incorporation of thymine can be related to incorporation atthe permissive temperature; however, incorporation of thymine can berelated to incorporation of uracil or of amino acids, all at therestrictive temperature. Similar incorporation (phenotype) pairs includelabelled uracil (inhibition of transcription); labelled amino acid(arrest of translation); labelled fatty acid precursor or fatty acid(arrest in membrane biosynthesis); D-alanine for bacteria or glucose ina short pulse for fungi (inhibition of cell wall synthesis).

Susceptibility to antibiotics is another important phenotype. It isuseful to obtain a "fingerprint" of resistance to a variety ofantibiotics at the permissive temperature and at various temperaturesapproaching the restrictive temperature. For example, if there ishypersusceptibility to rifampicin, the mutation likely affectstranscription. Hypersensitivity to several agents suggests a lesionwhich affects the cell envelope. Partial resistance to kanamycin may berelated to an alteration of the electrochemical gradient. A strain inwhich the ts lesion is complemented by the wild type DNA on a multi-copyplasmid may result in enhanced resistance to antibiotics when wild typeDNA is cloned. In E. coli, transferring a lesion into a secA-lacZindicator strain suggests blocking secretion. Changes in potassiumefflux in bacteria after shifting to the restrictive temperature,possibly in combination with kanamycin resistance, suggests analteration in the electrochemical gradient, such as collapse of electronpotential. Isolation and characterization of temperature-resistantpseudo-revertants of ts mutants suggests mutational suppression. Changesin gene expression at permissive and restrictive temperatures can beshown by 2-D gels, HPLC, or FPLC protein profiles. Total protein andpulse-labelled protein can be detected. Correlation of the changes witha pattern (e.g., shifting cells to medium containing a particularantibiotic) suggests a mutation affecting protein expression.

After isolation of ts mutants, complementation with wild type DNA, andoptional prioritization, the DNA sequence and homologies are determined.It is also desirable to identify redundant mutants prior to theircomplementation by libraries. Redundant ts mutants can be identified bytesting for the ability of complementation clones to complement tsmutations in other mutant strains. It is burdensome to test each plasmidthat complements one ts mutant for the ability to complement other tsmutants. Where electroporation is used (e.g., S. aureus), an efficientapproach can use plasmid pools containing 20 different plasmids. Largerplasmid pools are difficult to prepare due to low transformationfrequencies and the reversion frequencies of some ts mutants. It is alsodesirable to measure growth and microscopically inspect the cellsfollowing a temperature shift; perform uptake studies; and other variouselaborate studies.

H. Use

The invention features methods of rapidly identifying strains withconditional lethal mutations from a large group of mutants. In general,the gene and corresponding gene product are subsequently identified,although the strains themselves can be used in screening or diagnosticassays. The mechanism(s) of action for the identified genes and geneproducts provide a rational basis for the design of therapeutic agentswhich are, for example, lethal against a bacterium used in the methodand related species, rather than being merely bacteriostatic.

For example, where the organism used in the method is a fungal orbacterial species, therapeutic agents are antifungal or antibacterialagents. These agents reduce the action of the gene product in a wildtype strain, and therefore are useful in treating a subject with thattype, or a similarly susceptible type of infection by administering theagent to the subject in a pharmaceutically effective amount. Reductionin the action of the gene product includes competitive inhibition of thegene product for the active site of an enzyme or receptor;noncompetitive inhibition; disrupting an intracellular cascade pathwhich requires the gene product; binding to the gene product itself,before or after post-translational processing; and acting as a geneproduct mimetic, thereby down-regulating the activity. Therapeuticagents include monoclonal antibodies raised against the gene product.

Furthermore, the presence of the gene sequence in certain cells (e.g., apathogenic microbe of the same genus or similar species), and theabsence or divergence of the sequence in host cells can be determined.Therapeutic agents directed toward genes or gene products which are notpresent in the host have obvious advantages including fewer sideeffects, and lower overall dosage. Enzymatic or ligand binding activitycan also be assayed. Other uses include determination of targets foranti-parasitic drugs or insectides.

OTHER EMBODIMENTS

From the above description, the essential characteristics of the presentinvention can be easily ascertained. Without departing from the spiritand scope thereof, various changes and modifications of the inventioncan be made to adapt it to various usages and conditions. Thus, otherembodiments are also within the claims.

Examples of other embodiments include a device which performs at leaststeps (b) and (c), exposing the cells to restrictive conditions and thenshifting the cells to the second permissive conditions; and a devicewhich performs at least step (d), optically scanning, staining, orotherwise identifying and selecting cells which failed to grow whenshifted to the second permissive conditions.

What is claimed is:
 1. A method for identifying a strain carrying aconditional lethal mutation in a gene, the method comprising:(a) growingstrains under first permissive conditions; (b) exposing strains fromstep (a) to restrictive conditions for a period of time equivalent to atleast two growth cycles; (c) shifting the strains from step (b) tosecond permissive conditions for a period of time equivalent to at leastten growth cycles; and (d) selecting a strain that survives step (a) butdoes not survive steps (b) and (c), thereby identifying a straincarrying a lethal mutation that is sensitive to the restrictiveconditions and essential for survival of the strain.
 2. A method ofclaim 1, further comprising after step (d), the step of selecting astrain carrying a recessive conditional lethal mutation.
 3. A method ofclaim 2 in which the strains from step (a) are strains of Saccharomycescerevisiae.
 4. A method of claim 1, further comprising after step (d),the step of isolating from said strain a gene carrying said mutation. 5.A method of claim 4, further comprising, after the step of isolatingsaid gene, a step selected from the group consisting of identifying thefunction of said gene, identifying the product expressed by said gene,and sequencing said gene.
 6. A method of claim 1 in which the firstpermissive conditions include a complete medium for the strains.
 7. Amethod of claim 1 in which the first permissive conditions include amedium at low osmotic strength.
 8. A method of claim 1 in which thefirst permissive conditions include a temperature between 5° and 15° C.below the optimal growth temperature for the wildtype.
 9. A method ofclaim 1 in which the restrictive conditions include a temperaturebetween the optimal growth temperature for the strains and 15° C. abovethe optimal growth temperature for the strains.
 10. A method of claim 1in which the second permissive conditions are substantially the same asthe first permissive conditions.
 11. A method of claim 1 in which thestrains in growing step (a) are replica plated cells.
 12. A method ofclaim 1 in which the period of time in step (c) is equivalent to atleast fifteen growth cycles.
 13. A method of claim 1 in which thestrains from step (a) are selected from the group consisting ofbacteria, fungi, and yeast.
 14. A method of claim 13 in which thestrains from step (a) are strains of Escherichia coli, Streptococcuspneumoniae, or Staphylococcus aureus.
 15. A method of claim 13 in whichthe strains from step (a) are strains of Aspergillus nidulans.
 16. Amethod of claim 1 in which the strains from step (a) have beenmutagenized.
 17. A method of claim 16 in which the strains have beenmutagenized with a chemical mutagen selected from the group consistingof ethyl methanesulfonate, methyl methanesulfonate,methylnitrosoguanidine, 4-nitroquinoline-l-oxide, 2-aminopurine,5-bromouracil, ICR 191, acridine derivatives, ethidium bromide, nitrousacid, and N-methyl-N'-nitroso-N-nitroguanidine.